How oncogenes integrate metabolic pathways to meet the biosynthetic demands of cancer cell growth and proliferation is a central question of cancer research with broad clinical implications. In high-risk neuroblastoma, one of the deadliest childhood cancers, the mevalonate pathway is transcriptionally activated. This metabolic pathway uses acetyl-CoA carbon to produce cholesterol and other metabolites essential to sustain the proliferation of neuroblastoma cell lines in culture and the growth of neuroblastoma tumors in mouse models. We recently obtained evidence that the oncogenic transcription factor MYCN is required for the transcriptional activation of mevalonate pathway enzymes and the increased production of cholesterol in cell lines derived from high-risk neuroblastoma tumors with genomic amplification of MYCN. The proposed research will address two major questions concerning the molecular basis of MYCN action in reprogramming of mevalonate metabolism and cholesterol synthesis: 1) how MYCN activates the mevalonate pathway to increase its output; and 2) how MYCN coordinates other metabolic pathways to increase the supply of acetyl- CoA. In Aim 1, we will use a combination of cellular and molecular approaches to test the hypothesis that MYCN disrupts end-product feedback inhibition of the mevalonate pathway by transcriptional activation of SCAP, a positive regulator of mevalonate metabolism and repression of INSIG2, a negative regulator, leading to constitutive activation of the mevalonate pathway. We will further investigate the molecular basis for MYCN repression of INSIG2 expression. In Aim 2, we will use stable isotope tracers in combination with shRNA silencing and enzyme inhibitors to delineate the metabolic pathways that supply the substrate acetyl-CoA. We will test the hypothesis that MYCN increases the flux of glutamine carbon into the mevalonate pathway for cholesterol synthesis via transcriptional activation of nucleotide and serine-glycine synthesis pathways. In Aim 3, we will use stable isotope tracers in combination with enzyme inhibitors to provide in vivo evidence for glutamine as a major source of carbon for cholesterol synthesis via nucleotide and serine-glycine synthesis pathways in patient-derived xenografts and the TH-MYCN mouse model of high-risk neuroblastoma. Successful completion of this project will define a molecular mechanism for MYCN to integrate nucleotide, amino acid, and cholesterol metabolism in driving and sustaining high-risk neuroblastoma, which may suggest new avenues of therapeutic intervention.